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STUDIES OF THE INTERACTIONS OF TRANSPORTIN-SR2 WITH HIV-1 INTEGRASE AND RAN Oliver Taltynov (1), Jonas Demeulemeester (1), Stéphanie De Houwer (1), Wannes Thys (1), Stephen Weeks (2), Sergei Strelkov (2), Frauke Christ (1), Zeger Debyser (1) (1) Division of Molecular Medicine, Katholieke Universiteit Leuven, Leuven, 3000, Flanders; (2) Laboratory for Biocrystallography, Department of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, 3000, Flanders Background: Transportin-SR2 (TRN-SR2) is a nuclear import factor responsible for the transport of the HIV-1 preintegration complex (PIC) into the cell nucleus. This transport is likely conducted by the interaction between TRN-SR2 and HIV-1 integrase (IN). Alike most nuclear import proteins driving force of this specific import is a gradient of small GTPase Ran which in its GTP-bound form releases cargo (the integrase) in the nucleus from Transportin-SR2. Methods: We used gel filtration chromatography and AlphaScreen to characterize the complex formation of His-TRN-SR2 with RanGTP/GDP and HIV-1 IN. Small Angle X-Ray Scattering (SAXS) has been used to study His-TRN-SR2 and the stoichiometry of the purified complexes. Results: By gel filtration we demonstrate clearly the interaction between His-TRN-SR2 and both, RanGTP/GDP and HIV-1 IN deletion mutants (the catalytic core/C-terminal domain and the C-terminal domain). Alphascreen measurements confirm the tight interaction. As expected RanGTP binds with higher affinity to His-TRN-SR2 as its GDP counterpart. Remarkably, whereas the interaction of His-TRN-SR2 with HIV-1 IN His-catalytic core-C-terminal domain is causing expected shift to earlier elution of the complex during gel filtration, the interaction of His-TRN-SR2 with RanQ69L.GTP brings along the opposite. It points out that TRN-SR2 takes up more condensed structure while binding Ran. His-TRN-SR2 had been proven to be monomer in solution by SAXS measurements. By SAXS the stoichiometry of the interactions of His-TRN-SR2 with its binding partners have been determined.